Compositions and method for storage of nucleic acid from bodily fluids

Abstract

The present invention provides an aqueous composition comprising SDS, Cyclohexanediamine tetraacetate, Tris-HCl and proteinase K for the extraction of nucleic acid from a sample of bodily fluid, such a saliva, wherein the extracted nucleic acid is stable for at least fourteen days at room temperature The composition permits direct use of the extracted and stored DNA in an amplification reaction without further processing.

1 . An aqueous composition for extracting and preserving nucleic acid from a sample of a bodily fluid such that said nucleic acid within said sample remains stable for at least 14 days at room temperature following mixture with said aqueous composition, said aqueous composition comprising:
 (i) a denaturing agent   (ii) a chelator;   (iii) a buffering agent at a pH of about pH 7 to about pH 11; and   (iv) a protease;   wherein said composition does not inhibit nucleic acid amplification when said composition is added to an amplification reaction mixture at an amount of at least 2% (vol./vol.) of the total volume of said amplification reaction mixture, and wherein the final concentration of said denaturing agent in said amplification reaction mixture is less than about 0.01% (wt./vol.).   
     
     
         2 . The aqueous composition of  claim 1 , wherein the bodily fluid is saliva. 
     
     
         3 . The aqueous composition of  claim 1 , wherein said denaturing agent is SDS, said chelator is CDTA, said buffering agent is TrisHCl, and said protease is proteinase K. 
     
     
         4 . The aqueous composition of  claim 3 , which comprises 0.05% SDS, 20 mM CTDA, 200 mM TrisHCl pH 8.0, 400 mM NaOAc, and 10 μg/ml proteinase K. 
     
     
         5 - 9 . (canceled) 
     
     
         10 . The aqueous composition of  claim 1 , wherein said composition does not inhibit nucleic acid amplification when added to said amplification reaction mixture in an amount of between 2% (vol.) and 10% (vol.) of the total volume of the amplification reaction mixture. 
     
     
         11 . The aqueous composition of  claim 1 , wherein said nucleic acid remains stable at room temperature for at least three hundred and sixty-five days. 
     
     
         12 . A method of amplifying deoxyribonucleic acid (DNA) DNA directly from a bodily fluid, comprising:
 (a) mixing a sample of the bodily fluid with the aqueous composition of  claim 1 ; and   (b) diluting at least 2% (vol./vol.) of the mixture formed in step (a) in an amplification reaction mixture without prior extraction of nucleic acid molecules present in said mixture and subjecting said amplification reaction mixture to polymerase chain reaction (PCR) amplification, wherein said PCR amplifies said nucleic acid molecules.   
     
     
         13 . The method of  claim 12 , wherein the bodily fluid is saliva. 
     
     
         14 . The method of  claim 12 , wherein the mixture formed in step (a) is stored at room temperature for at least fourteen days prior to step (b). 
     
     
         15 . The method of  claim 12 , wherein the mixture formed in step (a) is stored at room temperature for at least three hundred and sixty-five days prior to step (b). 
     
     
         16 . The method of  claim 12 , additionally comprising the step of heating the mixture at a temperature of from about 45° C. to 80° C. for about 15 to 60 minutes prior to step (b). 
     
     
         17 . A kit for amplifying deoxyribonucleic acid (DNA) from a bodily fluid, comprising:
 (a) the aqueous composition of  claim 1 ; and   (b) instructions for the use thereof.   
     
     
         18 . The kit of  claim 17 , wherein said kit is capable of use with saliva as the bodily fluid. 
     
     
         19 . A method of amplifying deoxyribonucleic acid (DNA) directly from a bodily fluid, comprising:
 (a) obtaining a sample comprising a bodily fluid admixed with a composition comprising:
 (i) a denaturing agent; 
 (ii) a chelator; 
 (iii) a buffering agent that establishes a pH of said sample of about pH 7 to about pH 11; and 
 (iv) a protease, 
 wherein nucleic acid molecules within said sample remain stable for at least 14 days at room temperature; 
   (b) preparing an amplification reaction mixture comprising at least 2% (vol./vol.) of the sample, wherein said amplification reaction mixture is prepared without prior extraction of said nucleic acid molecules from said sample and the final concentration of said denaturing agent in said amplification reaction mixture is less than about 0.01% (wt./vol.); and   (c) subjecting said amplification reaction mixture to polymerase chain reaction (PCR) amplification, wherein said PCR amplifies said nucleic acid molecules.   
     
     
         20 . The method of  claim 19 , wherein the bodily fluid is saliva. 
     
     
         21 . The method of  claim 19 , wherein the sample is stored at room temperature for at least fourteen days prior to step (b). 
     
     
         22 . The method of  claim 19 , wherein the sample is stored at room temperature for at least three hundred and sixty-five days prior to step (b). 
     
     
         23 . The method of  claim 19 , additionally comprising the step of heating the sample at a temperature of from about 45° C. to 80° C. for about 15 to 60 minutes prior to step (b). 
     
     
         24 . The aqueous composition of  claim 1  further comprising said bodily fluid. 
     
     
         25 . The aqueous composition of  claim 24 , wherein said bodily fluid is saliva. 
     
     
         26 . The aqueous composition of  claim 1 , wherein said protease is added with, or after addition of, said bodily fluid.